Principal Investigator/Program Director (Last, first, middle): Karas, Richard, H. RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? l Yes m No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? l Yes m No IACUC Approval Date: Animal Welfare Assurance Number A3775-01 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 9702-ABSTRACT.pdf Mime Type: application/pdf 7. * Project Narrative 8054-Project_Narrative.pdf Mime Type: application/pdf 8. Bibliography &References Cited 565-Literature_Cited.pdf Mime Type: application/pdf 9. Facilities &Other Resources 6777-resources.pg1.pdf Mime Type: application/pdf 10. Equipment Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Karas, Richard, H. Estrogen receptors (ERs) mediate the diverse effects of estrogen on the cardiovascular system. The two known ERs, ER[unreadable] and ER[unreadable], are ligand-activated transcription factors. The transcriptional effects of ERs depend on receptor interacting proteins, including SRC3, a member of the family of co-activator proteins. Commonly, the genomic effects just described are differentiated from more rapid effects that do not require alterations in gene expression and thus are called `non-genomic'effects. The best-studied non-genomic ER pathway in vascular endothelial cells (EC) involves rapid activation of the kinase Akt, and subsequent activation of endothelial nitric oxide synthase (eNOS). eNOS protein abundance is also transcriptionally regulated by ERs. These two signaling pathways, genomic and non-genomic, have largely been considered independent, and the physiologic relevance of the non-genomic pathway has been questioned. We recently identified the protein striatin as an ER[unreadable] interacting protein necessary for assembly of cell membrane-associated signaling complexes that mediate non-genomic estrogen-induced eNOS phosphorylation. Initially, we demonstrated that inhibition of ER[unreadable]-striatin binding blocked rapid activation or Akt and eNOS, but did not inhibit transcriptional activation of a estrogen-response-element driven reporter, supporting that the striatin-ER[unreadable] interaction is not required for transcriptional activation by ER[unreadable]. However, we now report that inhibition of ER[unreadable]-striatin binding prevents ER[unreadable]-mediated transcriptional regulation of the eNOS gene, and that the co-activator protein SRC3 in necessary for both non-genomic and genomic regulation of eNOS by ER[unreadable]. Based on these findings, we propose the central hypothesis of this application, that a multi-protein complex that includes ER[unreadable], the scaffolding protein striatin, and the coactivator SRC3 regulates both non-genomic and genomic effects of estrogen in EC and vascular physiology. We propose to test this hypothesis with the following specific aims: Specific Aim 1: To investigate the mechanisms by which striatin and SRC3 regulate rapid (non-genomic) ER-dependent activation of eNOS in ECs, Specific Aim 2: To investigate the mechanisms by which striatin and SRC3 regulate ER-dependent transcriptional (genomic) activation of eNOS in ECs, and Specific Aim 3: To determine the importance of ER-striatin binding in vivo by studying a transgenic mouse that expresses a peptide that disrupts ER[unreadable]-striatin binding. The results of these studies will provide insight into the importance of non-genomic ER[unreadable]-mediated signaling in vascular cells and its role in regulating longer-term genomic effects of the receptor. These studies have important implications for furthering our understanding of the effects of estrogen on the vascular system and for developing novel therapeutic agents that act via these signaling pathways. Project Description Page 6